US5011780A - In vitro embryo culture technique - Google Patents
In vitro embryo culture technique Download PDFInfo
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- US5011780A US5011780A US07/208,366 US20836688A US5011780A US 5011780 A US5011780 A US 5011780A US 20836688 A US20836688 A US 20836688A US 5011780 A US5011780 A US 5011780A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
- C12M3/10—Tissue, human, animal or plant cell, or virus culture apparatus for culture in eggs
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/02—Breeding vertebrates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K45/00—Other aviculture appliances, e.g. devices for determining whether a bird is about to lay
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/10—Bioreactors or fermenters specially adapted for specific uses adapted for the cultivation of avian eggs or in avian eggs, e.g. for vaccine production
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/24—Gas permeable parts
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
Definitions
- This invention relates to an in vitro avian embryo culture technique, which is particularly suitable for application to poultry, particularly hens.
- the chick embryo in the initial stages of its development from fertilisation to cleavage, has not been amenable to experimental intervention on account of the bulk, fragility and relative inaccessibility of the ovum. This problem has been discussed in recent reviews on the possible routes of transfer of exogenous genes into birds (Freeman and Messer, 1985; Crittenden and Salter, 1986). Perry (1986a,b) has taken the alternative view and suggested that genetic manipulation of the avian ovum is practicable. The aim of devising a complete culture system for the chick embryo was to provide a means of rearing the manipulated ovum to maturity. A method has now been established for in vitro culture to an intermediate stage of embryonic development, and progress is being made on this account. The technique will have applications not only in the genetic engineering of poultry, but also in the investigation of fundamental mechanisms of avian development and in the study of deleterious traits. Moreover, it will afford a desirable alternative to surgical intervention in the laying hen.
- the chick embryo originates in the germinal disc, a small region of cytoplasm situated at the animal pole of the ovum (the familiar yolk). During the first third of its development, the embryo remains floating at the surface of the yolk whilst the extra-embryonic membranes grow around the yolk and become vascularised. In the remaining period of development, the embryo grows at the expense of the food reserves in the egg. For present purposes, the development of the chick has been divided into 3 phases according to the changing requirements at successive stages from fertilisation to hatch.
- Phase I Fertilisation to blastoderm formation. This phase takes place in the oviduct and terminates at oviposition. Gamete interaction occurs within 15 min after ovulation, and the first cleavage division some 4 h later (Perry, 1987). In the following 20 h, subsequent divisions give rise to a simple sheet of cells overlying a subblastodermal cavity (Kochav, Ginsburg and Eyal-Giladi, 1980). During its passage through the oviduct, the ovum is invested with albumen in the magnum, then with the shell membrane in the isthmus where cleavage commences.
- the albumen In the uterus, the albumen is doubled in volume by the absorption of uterine fluid (pumping fluid), and finally the shell undergoes slow calcification. For hens laying in long sequences of one egg per day, oviposition is followed within 15-30 min by the next ovulation and the cycle is repeated.
- Phase II Embryonic morphogenesis. This phase takes place in the first 3 days of incubation of the egg (stages 1-18, Hamburger and Hamilton [1951]). At stage 20, the embryo is 10 mm in length, and the extra embryonic blastoderm extends around the yolk to its equator.
- Phase III Embryonic growth. This phase takes place in the final 18 days of incubation of the egg (stages 18-45, Hamburger and Hamilton [1951]).
- the invention provides, in a first embodiment, a process for the in vitro culture of avian embryos during the embryonic growth phase, the process comprising incubating an embryo in a close container, there being an air space above the embryo, the air space being separated from the external atmosphere by a partially gas permeable seal.
- the container may be part of an egg, such as a hen egg, and the depth of the space between the embryo and the seal is preferably from about 5 to about 15 mm.
- the incubated embryo is preferably gently agitated, at least initially.
- a process for the in vitro culture of an avian embryo during embryonic morphogenesis, the process comprising incubating an embryo in a culture medium in a liquid-filled closed liquid-impermeable container.
- the container may be partially gas permeable, and the culture medium is preferably liquid albumen.
- the cultured embryos should be subjected to gentle to moderate agitation.
- a process for the in vitro culture of an avian embryo during embryonic morphogenesis and during the embryonic growth phase, the process comprising first culturing the embryo by a process in accordance with the second embodiment disclosed above and subsequently culturing the embryo by a process in accordance with the first embodiment disclosed above.
- a process for the in vitro culture of an avian embryo up to blastoderm formation, the process comprising culturing a fertilised ovum, having a surrounding capsule of dense albumen, partially submerged in culture medium.
- the medium should be generally in line with the germinal disc.
- a suitable culture medium is liquid albumen, which may be diluted by water and/or a salt solution. This process preferably takes place in a closed container.
- a process for the in vitro culture of an avian embryo up to blastoderm formation and during embryonic morphogeneiss, the process comprising culturing an avian embryo by a process in accordance with the fourth embodiment disclosed above and subsequently culturing the embryo by a process in accordance with the second embodiment disclosed above.
- the invention provides a process for the in vitro culture of an avian embryo up to blastoderm formation, during embryonic morphogenesis and during embryonic growth to hatch, the process comprising culturing an avian embryo by a process in accordance with the fourth embodiment disclosed above, subsequently culturing the embryo by a process in accordance with the second embodiment disclosed above, and subsequently culturing the embryo by a process in accordance with the first embodiment described above.
- FIG. 1 shows a scheme of culture systems for chick embryos to indicate the periods of development covered by individual and linked systems, or a time scale of 0 to 22 days and specifically shows the period of chick embryonic development covered by a series of three culture systems, and the time of transfer of the embryos between systems for complete development in culture;
- FIG. 2 illustrates the structure of the newly laid egg of the hen
- FIG. 3 is a diagram of a culture system for Phase III (day 4 to hatch);
- FIG. 4 is a diagram of a culture system for Phase II (day 1 to day 4 or day 9) and;
- FIG. 5 is a diagram of a culture system for Phase I (fertilised ovum to day 1).
- a process for the in vitro culture of avian embryos during the embryonic growth phase comprising incubating an embryo in a closed container, there being an air space above the embryo, the air space being separated from the external atmosphere by a partially gas permeable seal.
- the seal is preferably in the form of a film.
- the seal may be of plastics material, for example polyethylene. It has been found that commercially available cling-film forms an appropriate seal, particularly when used in two layers. Any other material having the appropriate properties of cling film may be used.
- the suitability of material for use as a seal may be measured indirectly by measuring carbon dioxide and/or water vapour permeability; carbon dioxide permeabiIIty can be measured by testing the rise in pH of egg albumen after 24 hours incubation at 38° C. in otherwise impermeable containers.
- the albumen should be initially gassed with carbon dioxide to lower the pH 0.1.
- a pH rise of from 0.5 to 1.5 is generally suitable, with the preferred range being 0.5 or 0.7 to 1.0 or 1.3, for example about 0.9
- Water vapour permeability may be from 5 or 10 to 30 or 40 mg/cm 2 /24 h.
- the container is preferably part of an egg, which will usually be chosen from the same species as is being cultured. It has been found particularly appropriate to remove the blunt end from a whole egg; a 40 mm diameter hole centered on the axis of the egg was found to be particularly suitable.
- the hole at the blunt end of the egg is sealed with the partially gas permeable seal.
- the seal may be made to adhere to the egg shell by means of albumen.
- the permeability characteristics are similar to those of a natural egg.
- Culture medium may be present in some embodiments of this aspect of the invention, particularly if prior culture stages have been in vitro, but the process can work without it, for example when the prior culture stages have been carried out naturally.
- the culture medium when present will usually comprise albumen, either undiluted or in a dilute form and desirable inter-uterine fluid.
- the depth of the space between-the embryo and the seal is from 5 to 15 mm, for example about 10 mm.
- incubated embryo it is preferred to agitate the incubated embryo gently, at least initially.
- gentle agitation can be achieved by intermittent rocking, for example through an angle at 30°.
- Conventional incubation temperatures for example about 38° C. may be maintained.
- In vitro culture processes in accordance with this aspect of the invention are suitable for use from about day 4 (counting from fertilisation) up to hatch, which generally occurs at about day 22. However, for the final few days (for example 13) of embryonic life, it is preferred that there be no agitation.
- the seal be perforated to allow a certain amount of air into the container. Further air may be allowed in later, for example by removing the seal and optionally covering the hole in the eggshell (when the egg forms the container) by a solid disc, which may be provided by a petri dish.
- a process for the in vitro culture of an avian embryo during embryonic morphogenesis comprising incubating an embryo in a culture medium in a liquid-filled closed liquid-impermeable container.
- the container may be partially gas permeable.
- the gas permeability may be provided by an eggshell (generally in combination with the inner shell membrane) and/or by an otherwise partially gas permeable seal, whose preferred characteristics are as for the first aspect of the invention. It should be noted that the eggshell and inner shell membrane are partially gas permeable.
- the culture medium is preferably liquid albumen, which may be collected from freshly laid eggs.
- the container is for preference again part of an egg, but the preferred construction is somewhat different from that of the first aspect of the invention.
- it is preferably the sharp end which is removed, for example by a 32 mm hole. This ensures the presence of an air space between the outer shell membrane and the inner shell membrane; this appears to be advantageous as the air space expands during culturing to make up for the water lost by evaporation.
- the egg may be cultured in a generally horizontal position, with the seal then being to one side.
- the seal should be kept firmly in place against the shell of the egg.
- the cultured embryos be subjected to gentle to moderate agitation. Intermittent or continuous rocking, for example through an angle of 90° in hourly cycles, or other comparable intervals, is preferred.
- a process in accordance with this aspect of the invention will generally begin at about 1 day after fertilisation (that is to say about the normal time of laying) and may last for 2 or more days, for example up to 8. However, it is preferred that a process in accordance with this aspect of the invention only be continued for in the order of 3 or 4 days before transferring the embryo to a process in accordance with the first aspect.
- a process for the in vitro culture of an avian embryo during embryonic morphogenesis and during the embryonic growth phase comprising first culturing the embryo by a process in accordance with the second aspect of the invention and subsequently culturing the embryo by a process in accordance with the first aspect.
- transition occurs between 2 and 5, for example 4, days after fertilisation.
- a process for the in vitro culture of an avian embryo up to blastoderm formation comprising culturing a fertilised ovum, having a surrounding capsule of dense albumen, partially submerged in culture medium.
- the medium should be generally in line with the germinal disc, which will generally be uppermost, but below the level of the albumen capsule.
- the fertilized egg may be obtained surgically from the hen. If surgical techniques are used, the egg is preferably taken from the mid magnum, for example from 50 to 150 mm from the isthmus. Taking a fertilized egg from this area of the magnum has been found to be advantageous in that the fertilized ovum then appears to have an optimum thickness of the surrounding capsule of dense albumen.
- the culture medium can be liquid albumen which may be diluted by water and/or a salt solution. It is generally preferred to initiate culture with dilute albumen (for example 3:2) with salt solution and subsequently (for example after 1 day) to dilute the albumen with salt solution (for example 2:1).
- a process in accordance with this aspect of the invention preferably takes place in a closed container.
- the container may be made of an impermeable material such as glass, which may be sealed with a low gas permeability film such as SARAN WRAP (trade mark). Any other material having the appropriate properties of SARAN WRAP may be used.
- Gas permeability may be measured (eg for carbon dioxide and/or water vapour) as described above.
- the 24 hour pH rise may be from 0.5 to 1.0, for example from 0.6 to 0.8 in the carbon dioxide permeability test.
- the water vapour permeabiIity may be from 1.0 to 10, for example 2 to 5 mg/cm 2/24 h.
- an embryo cultured in accordance with this aspect of the invention will subsequently be cultured in accordance with a process of the second aspect of the invention. Therefore, according to a fifth aspect of the invention, there is provided a process for the in vitro culture of an avian embryo up to blastoderm formation and during embryonic morphogenesis, the process comprising culturing an avian embryo by means of a process in accordance with the fourth aspect of the invention and subsequently culturing the embryo by a process in accordance with the second aspect.
- a process for the in vitro culture of an avian embryo up to blastoderm formation, during embryonic morphogenesis and during embryonic growth to hatch comprising culturing an avian embryo by a process in accordance with the fourth aspect of the present invention, subsequently culturing the embryo by a process in accordance with the second aspect of the culturing invention and subsequently the embryo by a process in accordance with the first aspect of the invention.
- the recipient eggs, if eggs are used as containers, in the second stage are slightly larger than the donor eggs, for example by an amount of to 2 ml. If eggs are used as the containers in the third stage, the recipient eggs are preferably substantially (for example about 18 ml) larger than that used in the immediately preceding stage.
- Fertility was regularly checked by visual inspection of unincubated eggs and found to be better than 90%. In fertile eggs, the germinal region is seen as a white ring (3-4 mm in diameter) enclosing a semi-transparent area, and in infertile eggs it appears as a vaculoated disc (2-3 mm in diameter).
- Eggs Eggs laid in the preceding 24 h by the laying stock were used as a source of embryos for culture systems II and III (Examples 1-3). Freshly laid eggs (fertile and infertile) from this stock were also used as a source of albumen for the culture medium. Liquid albumen was collected from the inner and outer albumen layers of the egg (FIG. 2) and used within the same day for culturing. Recipient shells for culture system II (Examples 2-5) were obtained from the laying stock. The larger shells used as culture vessels for system III (Examples 1, 3, 5) were from double-yolked eggs of a commercial broiler strain obtained from a local hatchery (D.B. Marshall, Whitburn, West Lothian) and used for culture within 1-2 weeks of lay.
- a commercial broiler strain obtained from a local hatchery (D.B. Marshall, Whitburn, West Lothian) and used for culture within 1-2 weeks of lay.
- the culture medium was initially 30 gassed with CO 2 to lower the pH to an appropriate value.
- SARAN WRAP one layer
- the pH rose by an average of 0.7 unit, from pH 7.2-7.5 to pH 7.8-8.2, during incubation for 24 h at 41.5° C. and zero humidity.
- For cling film (2 layers) the pH rose by an average of 1.0 unit, from pH 7.2-7.5 to pH 8.3-8.5, during incubation for 24 h at 38° C. and R.H. 45-55%.
- Permeability to water vapour was determined by measuring the water loss from dishes (350 ml; diameter, 104 mm) containing 150 ml water. The dishes were sealed with wrapping film and incubated as described previously.
- the average permeability was 3.4 mg/cm 2 /24 hr (range, 3.203.8 mg).
- the average permeability was 22 mg/cm 2 /24 hr (range, 15-28 mg).
- the machines were cleaned and disinfected monthly with MILTON sterilising fluid (Richardson-Vicks Ltd). Sterility. All operations were conducted in semi-sterile conditions. The bacteriostatic properties of egg albumen made it unnecessary to take stringent aseptic precautions. Eggs were briefly rinsed in 70% alcohol shortly after collection, and then swabbed with 70% alcohol immediately before use. All equipment, distilled water and saline solutions were autoclaved. The salt solutions were filter sterilised. For the wrapping film, the outer layers of the roll of film were discarded, then sheets (100 mm 2 ) were cut and placed between sheets of sterile paper. This operation, collection of egg albumen and preparation of the cultures were performed in clean air cabinets. Antibiotics (penicillin, 100 u/ml; streptomycin, 100 mcg/ml) were added to the egg albumen which was used to glue the cling film closure to the recipient shells in system III.
- Recipient shells were prepared from double-yolked eggs. A circle of 40 mm in diameter was drilled around the blunt end of the egg and the cap of the shell, containing the air cell, was removed. After discarding the contents, the shell was rinsed out with distilled water, then refilled with water to prevent dehydration of the inner shell membrane. The volumes of the recipient shells ranged from 65 to 75 ml. Three-day incubated eggs, containing embryos at stages 15-20, were cracked open and the contents lowered into a shallow dish lined with cling film. The embryos were transferred in the cling film sac to the recipient shells, then the cling film was slowly withdrawn, keeping the embryos uppermost.
- the cultures were incubated at 38° C. and rocked intermittently through an angle of 30° for 5 days, then they were maintained in a stationary position for 10 days. For the final 3-4 days, they were placed in a stationary hatching incubator at 37° C. The relative humidity ranged from 45-60%. At 1-2 days before the estimated time of hatch, when the beak had pipped the chorioalloantoic membrane and penetrated the air space, small perforations were made in the cling film. The cling film was replaced with a petri dish lid several hours before hatch.
- the hatch rate was 36% (Table 2), and 72% of the hatchings appeared healthy. Their average weight was 35 g as compared with 46 g for control chicks grown in ovo. Abnormalities of the weaklings included incompletely retracted yolk sacs, unhealed navels and limb defects. Sticky chicks were common, the condition being associated with the presence of some unabsorbed albumen in the shell.
- Cling film seals for the recipient shells were used in place of the loose fitting lids employed by other workers.
- the cling film may be effective in regulating the environment in the space above the embryo by restricting gas exchange and water loss from this compartment.
- the shells were filled to the brim with liquid albumen (1-5 ml) collected from freshly laid eggs, and the aperture sealed with a sheet of cling film, taking care to avoid the inclusion of air bubbles in the preparation.
- the cling film was held in position by 2 rings (nylon) placed over either end of the shell and secured by elastic bands hooked 0 over a set of small pegs (FIG. 4).
- the reconstituted eggs were incubated on their sides, and rocked, intermittently or continuously, through an angle of 90° hourly cycles at 38° C. and relative humidity of 30-50%.
- the cultures were candled at 7, 9 and 10 days of incubation, and the surviving embryo reincubated.
- Example 2 Embryos from unincubated eggs were cultured for 3 days as described in Example 2 (FIG. 4). The preparations were then removed from the incubator, one at a time, an incision made in the shell and the contents transferred to a larger shell as described in Example 1 (FIG. 3) Attempts to remove the contents of the smaller shell through the existing aperture invariably damaged the embryo and the extra-embryonic membranes. The cultures were incubated as described in Example 1.
- Hens were taken at 2.75 h after the estimated time of lay, having ascertained that the laid egg was fertile. At this time in the daily reproductive cycle the next ovum has ovulated and is traversing the magnum where the albumen is deposited.
- the hens were killed with an intravenous injection of sodium pentobarbitone (EXPIRAL, Ceva Ltd.), the abdomen was plucked and swabbed with 70% alcohol. The portion of the oviduct containing the fertilised ovum was lifted out of the abdominal cavity and, after excision, it was placed in a sterile basin containing paper moistened with saline. Most of the ova were located at a distance of 50-150 mm from the border of the magnum with the isthmus.
- the total volume of medium required depended on the size of the yolk plus albumen capsule and amounted to 8-12 ml.
- the containers were tilted to ensure that the albumen capsule above the germinal disc was not submerged in medium.
- the preparations were incubated at 41°-42° C. for 24 h, with a delay of no more than 20 min between retrieval of the ovum from the magnum and incubation.
- the culture medium consisted of liquid albumen (2 parts), collected from the inner and outer albumen layers of freshly laid eggs, and salt solution part)
- the salt solution contained 50mM KHCO 3 , 30 mM NaHCO 3 , 10 mM KCl, 2.5 mM MgCl 2 .6 H 2 O, 0.7mM CaCl 2 .2 H 2 O and 11 mM glucose.
- the pH of the culture medium was lowered from an initial value of 8.4 to 7.2-7.4 by stirring in an atmosphere of CO 2 , and was maintained at the lower value during the course of the preparative procedures by storing the medium in sealed containers.
- the state of development after 24 h can be roughly ascertained by visual inspection of the germinal area. Growth of a blastoderm and formation of a subblastodermal cavity is indicated by an opaque ring (3 mm in diameter) enclosing a semi-transparent area However, for an accura.te assessment of developmental potential, the preparations were cultured for a further 3-6 days in system II.
- Embryos were placed in the culture system for Phase II as described in Example 2.
- Recipient shells were prepared from eggs that were about 3-4 g heavier than the preceding eggs laid by the donor hens.
- the cultures were cooled to ambient temperature and placed in the recipient shells which were then filled with the culture medium (10-20 ml) and sealed with cling film (FIG. 4).
- the reconstituted eggs were incubated on their sides at 38° C., R.H.30-50%, with intermittent rocking through an angle of 90° in hourly cycles.
- the cultures were removed from the shells for examination at 7, 8 or 9 days of incubation.
- the culture system for Phase I was based on a series of experiments designed to test the requirements for the first 24 h of development. Tests were made on ova at different stages after fertilisation and subjected to different treatments, before transfer to the standard culture system for Phase II for further development and analysis. Aspects of the culture system that were examined were: the depth of material overlying the germinal disc, the composition of the culture medium, gaseous exchange and the spatial position of the germinal disc.
- a precisely defined culture medium was not essential for development in Phase I. Embryos grew well in undiluted albumen for 6 days to Stages 25-26. However, for development beyond this stage it was necessary to use diluted albumen (Table 7).
- the composition of the salt solution was based on data given by Beadle, Conrad and Scott (1938) and Leonard (1968) for differences in ionic composition of the albumen of uterine eggs, prior to plumping, and of the albumen of laid eggs.
- Glucose was added at a concentration equivalent to that in uterine fluid (Davidson and Draper, 1969) at the commencement of the uterine phase of egg formation.
- the total amount of fluid (salt solution plus plumping fluid) was roughly similar to the amount of plumping fluid in the laid egg.
- Plumping fluid comprises about half of the albumen content of the egg, which varies from 36 to 40 ml between eggs.
- Experiments in which the albumen in the medium was diluted solely with distilled water gave similar results to those obtained using salt solution as a diluent (Table 7).
- salt solution as a diluent
- the pH of the medium was routinely adjusted to 7.2-7.4, with CO 2 to conform with the in vivo conditions; the oxygen level of the medium was assumed to be ambient.
- the vessels were sealed with WRAP to maintain the pH below 7.8, and to maintain humid atmosphere in the chamber.
- Kochav and Eyal-Giladi (1971) have grown uterine embryos from the multicellular stage to the 4-somite stage (St. 8).
- the shell membrane was removed, the yolk and albumen transferred to a beaker of physiological salt solution, and the yolk suspended by the chalaza to force the germinal area into a oblique position.
- the culture method for the development of the fertilised ovum for a period of 7 days provides a model system for testing the effects of experimental intervention at precleavage stages on a range of developmental processes in birds.
- Ova recovered from the mid-magnum region of the oviduct were cultured for 24 h in the system for Phase I (FIG. 5), transferred to the system for Phase II (FIG. 4) and incubated for 3 days.
- the procedures are described in Example 4, with the modification that the proportions of liquid albumen to salt solution in the culture medium were 3:2 for system I and 2:1 for system II.
- the recipient shells prepared for system II were 1-2 ml larger in volume than those of the preceding eggs laid by the donor hens. After a total of 4 days, incubation, the embryos were transferred to the system for Phase III (FIG. 3) as described in Example 3.
- the recipient shells prepared for system III were, on average, 18 ml larger in volume than the recipient shells used in system II.
- the volume difference determined the size of the airspace in the chamber.
- the cultures were incubated at 38° C. for 5 days, turning them through an angle of 30°, then for 10 days in a stationary position, and finally placed in a stationary hatching incubator at 36-37° C. (Table 8).
- the establishment of a complete in vitro system for the chick embryo has a wide range of potential applications in areas of basic and applied research. It provides the opportunity to manipulate the avian ovum by, for example, the injection of foreign genes or whole genomes, and to investigate the effects of such manipulations in the hatched chick and probably in the mature bird.
- the methods also give guidelines for devising in vitro techniques for oviductal embryos at earlier and more advanced stages than those presently employed, with applications in the fields of in vitro fertilisation and the insertion of putative totipotent cells into the embryo.
- a potential application is in the production of transgenic poultry. Modifications of the factors for growth and reproductive performance will be of benefit to the poultry industry. Furthermore, the insertion of genes for novel proteins into the avian germ line is a potentially valuable technique for the production of biomedically important proteins in egg white.
- the high reproductive capacity of the domestic fowl gives it an advantage over other farm animals in this technological field. A hen matures in 6 months and is capable of producing some 300 eggs in the first year of lay.
- the exemplified method for Phase III is a discrete system (Example 1).
- the exemplified method for Phase II overlaps that for Phase III (Example 2), but transfer between these systems should for preference be made at a specific stage due to the increased fragility of the embryo with age (Example 3).
- the method for Phase I covers a period commencing some 2 h after ovulation, when albumen deposition is underway and the male and female pronuclei are undergoing enlargement (Perry, 1987). It requires linkage with the method for Phase II for its analysis (Example 4) and, subsequently, with the method for Phase III for a complete culture system (Example 5).
Abstract
Description
TABLE 1 ______________________________________ Survival rates of chick embryos grown for specified periods in culture systems appropriate for particular phases of development from 2 h post- fetilisation (day 0) No. of Period of Culture embryos No. of surviving development systems cultured embryos or chicks (%) ______________________________________Day 4 to hatch III 69 25 (36)Day 1 today 8 II 47 35 (74)Day 1 to hatch II to III 59 16 (27)Day 0 to day 7 I to II 35 23 (67)Day 0 to hatch I to II to III 96 8 (8) ______________________________________
TABLE 2 ______________________________________ Survival rates of chick embryos at intervals after transfer to recipient shells on the third day of incubation in ovo. Culture system III. Days of incubation Number of Live embryos/ from lay live embryos total cultures (%) ______________________________________ 3 69 9 48 69 14 39 57 18 32 46 21 27 39 Hatched 21-22 .sup. 25.sup.+ 36 ______________________________________ .sup.+ The number of hatched chicks includes healthy chicks and weaklings
TABLE 3 ______________________________________ Survival rates of chick embryos at intervals after transfer from unincubated eggs to recipient shells. Culture system II Days of incubation Number of Live embryos/ from lay live embryos total cultures (%) ______________________________________ 0 47 6 39 83 7 35 74 9 15 32 ______________________________________
TABLE 4 ______________________________________ Survival rates of chick embryos at intervals after transfer from unincubated eggs to small recipient shells, and after 3 days, to large recipient shells. Culture systems II to III. Days of incubation Number of Live embryos/ from lay live embryos total cultures (%) ______________________________________ 0 59 3 55 93 9 34 58 14 28 47 18 21 36 21 17 29 Hatched 21-22 .sup. 16.sup.+ 27 ______________________________________ .sup.+ The number of hatched chicks includes healthy birds and weaklings
TABLE 5 ______________________________________ Development of fertilised chick ova recovered from the magnum at 2.75 h after the preceding eggs were laid. Culture systems I to II. Viability of embryos on Number Number of embryos given days of incubation of ova at Stages 27-29 7 8 9 ______________________________________ 35 24 2/2 9/17 0/4 ______________________________________
TABLE 6 ______________________________________ Development of fertilised chick ova cultured in recipient shells (system II) throughout incubation. Phase I culture system trial.sup.+. Percentage of cultures Incu- Blasto- Number bation Viable Malformed dermal Germinal of ova (days) embryos* embryos growth discs ______________________________________ 48 3-5 19 6 46 29 ______________________________________ .sup.+ For the first 24 h, the reconstituted eggs were placed in sealed plastic bags and rocked through an angle of 90° in hourly cycles. Thereafter they were incubated by the method for system II. *The embryos were retarded in developmental age by 12 h or more
TABLE 7 __________________________________________________________________________ Development of fertilised chick ova in culture media consisting of liquid albumen, undiluted, or diluted with salt solution or water alone. Phase I to II culture system trials. Percentage of cultures Number Incubation Normal Malformed Blastodermal Germinal Medium of ova (days) embryos.sup.+ embryos growth discs __________________________________________________________________________ Undiluted 35* 6-7 51 17 29 3 Diluted: salt solution 90* 7-8 51 13 24 12 water 17 7-8 59 18 0 23 __________________________________________________________________________ .sup.+ Mortality was high at 7 days' incubation in undiluted albumen, and 8 days' incubation in diluted albumen. *In these preparations, the culture vessels for the Phase I system were sealed with cling film and a layer of cling film was placed directly over the albumen capsule
TABLE 8 ______________________________________ Procedure for incubation of chick embryos from fertilised ovum to hatch Temper- Relative Successive days Rocked/ ature humidity (%) of incubation stationary Angle (°C.) in incubator ______________________________________ 0-1 Stationary -- 41-42 0 1-4 Rocked 90° 38 30-50 4-9 Rocked 30° 38 30-50 9-19 Stationary -- 38 40-55 19-22 Stationary -- 36-37 40-60 ______________________________________
TABLE 9 ______________________________________ Survival and hatch rates of fertilised chick ova at intervals in culture. Culture systems I to II to III. Days of Number of Live embryos/ incubation live embryos total cultures (%) ______________________________________ 0 96 4 55 57 10 31 32 15 26 27 19 18 19 22 10 10 Hatched 22-23 8.sup.+ 8 ______________________________________ .sup.+ Five chicks were healthy
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US5286641A (en) * | 1991-04-30 | 1994-02-15 | Director General, National Institute of Animal Industry | In vitro culture method for a fertilized ovum of a hen |
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GB8910933D0 (en) * | 1989-05-12 | 1989-06-28 | Agricultural & Food Res | In vitro embryo culture technique |
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-
1987
- 1987-06-19 GB GB878714426A patent/GB8714426D0/en active Pending
-
1988
- 1988-06-13 ZA ZA884225A patent/ZA884225B/en unknown
- 1988-06-16 IL IL86770A patent/IL86770A/en not_active IP Right Cessation
- 1988-06-17 FI FI882923A patent/FI94772C/en active IP Right Grant
- 1988-06-17 NZ NZ225076A patent/NZ225076A/en unknown
- 1988-06-17 DK DK198803353A patent/DK175847B1/en not_active IP Right Cessation
- 1988-06-17 US US07/208,366 patent/US5011780A/en not_active Expired - Lifetime
- 1988-06-17 NO NO882707A patent/NO882707L/en unknown
- 1988-06-17 PT PT87756A patent/PT87756B/en not_active IP Right Cessation
- 1988-06-17 IE IE184788A patent/IE62743B1/en not_active IP Right Cessation
- 1988-06-17 CA CA000569779A patent/CA1324972C/en not_active Expired - Lifetime
- 1988-06-18 KR KR1019880007351A patent/KR970000596B1/en not_active IP Right Cessation
- 1988-06-20 EP EP88305596A patent/EP0295964B1/en not_active Expired - Lifetime
- 1988-06-20 DE DE88305596T patent/DE3886274T2/en not_active Expired - Lifetime
- 1988-06-20 JP JP63150375A patent/JP2718946B2/en not_active Expired - Lifetime
- 1988-06-20 AT AT88305596T patent/ATE98423T1/en not_active IP Right Cessation
- 1988-06-20 ES ES88305596T patent/ES2047548T3/en not_active Expired - Lifetime
- 1988-06-20 AU AU18161/88A patent/AU1816188A/en not_active Abandoned
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IE62743B1 (en) | 1995-02-22 |
FI882923A0 (en) | 1988-06-17 |
IL86770A0 (en) | 1988-11-30 |
ZA884225B (en) | 1990-02-28 |
DK335388D0 (en) | 1988-06-17 |
AU1816188A (en) | 1988-12-22 |
PT87756B (en) | 1992-10-30 |
GB8714426D0 (en) | 1987-07-22 |
DK335388A (en) | 1988-12-20 |
DE3886274D1 (en) | 1994-01-27 |
JPH0198433A (en) | 1989-04-17 |
IE881847L (en) | 1988-12-19 |
ES2047548T3 (en) | 1994-03-01 |
PT87756A (en) | 1988-07-01 |
JP2718946B2 (en) | 1998-02-25 |
FI94772B (en) | 1995-07-14 |
FI882923A (en) | 1988-12-20 |
KR970000596B1 (en) | 1997-01-14 |
IL86770A (en) | 1993-01-31 |
NO882707D0 (en) | 1988-06-17 |
ATE98423T1 (en) | 1994-01-15 |
DK175847B1 (en) | 2005-03-29 |
NO882707L (en) | 1988-12-20 |
KR890000658A (en) | 1989-03-16 |
FI94772C (en) | 1995-10-25 |
EP0295964B1 (en) | 1993-12-15 |
DE3886274T2 (en) | 1994-04-14 |
NZ225076A (en) | 1990-11-27 |
EP0295964A1 (en) | 1988-12-21 |
CA1324972C (en) | 1993-12-07 |
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